Serological test for the detection of hepatitis

ABSTRACT

A diagnostic test procedure for infectious and serum hepatitis involves the cryogenic fractionation of human blood serum followed by careful separation of the bottom fraction of cryoprecipitate. A selected glycine-saline processing reagent having a particular concentration and pH is added to the fraction after separation thereof and the resultant mixture is incubated to provide a processed serum for both preliminary and confirmatory testing. In the preliminary test procedure equal portions of the processed serum and a special immunological diagnostic reagent are mixed on a glass slide. A positive reaction occurs within 1 minute as evidenced by the presence of a visible colloidal aggregate. The confirmatory test procedure utilizes a similar though more refined and discriminatory technique employing more precisely measured amounts of the processed serum and diagnostic reagent followed by intermixing and macroscopic reading of the sedimentation rate within the mixture. The immunological diagnostic reagent is a latex suspension in a saline buffer solution. The suspension contains polystyrene particles coated with a globulin fraction.

United States Patent McCuller SEROLOGICAL TEST FOR THE DETECTION OF HEPATITIS [72] lnventor: James F. McCuller, East Northport,

L.l., NY.

[73] Assignees: Leslie 1. Lukash, Rockville Center; George Shearer, Levittown; Joseph L. Scalise, Seaford, N.Y. part interest to each [22] Filed: Dec. 8, 1969 [21] Appl. No.: 883,261

52 u.s.c1. ..424/l2,260/ll2B, 260/122, 424/78, 424/86, 424/89 511 Int. Cl. ..A23j l/06,CO7g7/00,G01n33/l6 581 Field of Search ..424/11, 12, 13, 78, 81,89, 424/101; 260/112, 121, 122

[56] References Cited UNITED STATES PATENTS 3,297,532 1/1967 Jones ..424/85 X 3,088,875 5/1963 Fisk ..424/l2 3,461 ,200 8/ l 969 Mathies ..424/88 OTHER PUBLICATIONS Pike, AJCP, Vol. 30, July 1958, pp. 28- 31.

Kabat, Exptl. lmmunochem, C. C. Thomas Pub., Springfield, "1., 2nd Ed, l96l, pp. I24- I25, 638- 639.

Dresner, The N.E..l. Med., Vol. 261, Nov. 12, l959, pp. 981- 988.

[4 1 Oct. 10, 1972 Chem. Abs, Vol. 65, 1966, p. l4246f.

Chem. Abs., Vol. 69, pg. 9410, No. 9408w.

Balazs, Amer. J. Med. Sci., Vol. 245, 1963, pp. 206- 209.

Primary Examiner-Albert T. Meyers Assistant Examiner-A. P. Fagelson Att0rneyPrutzman, Hayes, Kalb & Chilton [57] ABSTRACT A diagnostic test procedure for infectious and serum hepatitis involves the cryogenic fractionation of human blood serum followed by careful separation of the bottom fraction of cryoprecipitate. A selected glycine-saline processing reagent having a particular concentration and pH is added to the fraction after separation thereof and the resultant mixture is incubated to provide a processed serum for both preliminary and confirmatory testing. In the preliminary test procedure equal portions of the processed serum and a special immunological diagnostic reagent are mixed on a glass slide. A positive reaction occurs within l minute as evidenced by the presence of a visible colloidal aggregate. The confirmatory test procedure utilizes a similar though more refined and discriminatory technique employing more precisely measured amounts of the processed serum and diagnostic reagent followed by intermixing and macroscopic reading of the sedimentation rate within the mixture. The immunological diagnostic reagent is a latex suspension in a saline buffer solution. The suspension contains polystyrene particles coated with a globulin fraction.

10 Claims, No Drawings SEROLOGICAL TEST FOR THE DETECTION OF HEPATITIS BACKGROUND AND SUMMARY OF THE INVENTION The present invention relates to the detection of hepatitis. More particularly, it is concerned with a new and improved diagnostic procedure for the selective detection of hepatitis including a serological test and the test reagents used therein.

Although the occurrence and transmission of hepatitis is relatively common, its etiology has not been fully documented or isolated. The disease is generally believed to be viral in nature and can be transmitted through polluted drinking water, raw shell fish, poor sanitary conditions or by blood transfusions. When it is transferred by transfusion, it is commonly designated serum hepatitis" rather than epidemic or "infectious hepatitis," although the two types of hepatitis are very closely related. Accordingly, unless otherwise specified, the term hepatitis" as used herein is intended to mean both infectious and serum hepatitis.

For many years investigators have attempted to gain a more complete understanding of the pathogenetic role of the immunologic processes in a liver damaged by hepatitis. Although the results of these investigations have led to many unproven theories, it has been established that the concentration levels of serum immunoglobulins generally change in the case of liver diseases with elevated concentrations being found in cases of hepatitis, cirrhosis and collagen diseases. This increased immunoglobulin concentration can be detected by the large number of globulin-forming cells in the spleen of patients with hepatitis and other liver diseases. Unfortunately the elevation of globulin concentration, and particularly gamma globulin, is not dependent on the specific cause of the liver disease so that the exact nature of the disease cannot be determined merely by detecting an increase in the number of globulin-forming cells. In recent years some specificity has evolved and it is now believed that gamma A globulins are elevated where the liver disease is hepatitis, cirrhosis, rheumatoid arthritis or the collagen diseases, and that gamma M globulins are increased in other liver diseases such as biliary cirrhosis.

It is also known that patients with hepatitis and other liver diseases frequently evidence a high incidence of false positive serologic tests for other conditions. For example, there is a frequent incidence of false positive serologic tests for syphilis in cases of acute hepatitis, cirrhosis and biliary cirrhosis, while false positive serologic reactions for rheumatoid factor are found in many cases of hepatitis.

Accordingly, it is a primary object of the present invention to provide a reliable means for selectively detecting infectious and serum hepatitis Another object of the present invention is to provide a new and improved immunological serologic test procedure and test reagents for the detection and diagnosis of both serum and infectious hepatitis. Included in this object is the provision for a specific diagnostic test procedure which allows the clinician to differentiate between hepatitis and other liver diseases.

A further object of the present invention is to provide a new and improved diagnostic test procedure and test reagents which not only permit the aforementioned differentiation between hepatitis and other liver diseases but also enable the clinician to make this differentiation in a rapid and reproducible manner without performing painful liver biopsies or large numbers of time-consuming liver chemistries, thus effecting a substantial saving of both time and expense.

An additional object of the present invention is to provide a method of concentrating and detecting a specific factor indigenous to serum hepatitis and infectious hepatitis thereby enabling a rapid yet reliable diagnosis.

Still another object of the present invention is to provide a new and improved test procedure which obviates the high percentage of nonspecific reactions of a false negative or false positive nature caused not only by liver diseases other than hepatitis but also by other factors which might be present in blood samples from normal persons, such as hemolyzed blood, sera containing red blood cell stroma or blood from patients having recently undergone extracorporeal circulatory procedures or having recently received cortisone therapy.

Another object of the present invention is to provide a highly selective and reliable test procedure for the detection of hepatitis which is also capable of distinguishing between infectious and serum hepatitis.

A still further object of the present invention is to provide a new and improved diagnostic test procedure and test reagents of the type described which permit blood banks and manufacturers of blood products to screen their blood donors in a simple and effective manner and to detect those donors who are carriers of hepatitis, thereby substantially reducing if not completely eliminating the transmission of hepatitis through the transfusion of blood or blood products.

Still another object of the present invention is to provide a diagnostic test procedure for hepatitis which is effective to produce positive test results from one month before the onset of the disease up to about twelve months after.

Other objects will be in part obvious and in part pointed out hereinafter.

These and related objects are accomplished in accordance with the present invention by providing a diagnostic test procedure which involves the cryogenic fractionation of human blood serum to be tested to ef fect concentration and separation of a hepatitis factor within a cryogenic fraction followed by the subsequent detection of that factor with a suitable immunological diagnostic reagent to provide a demonstrative indication of the presence ofinfectious or serum hepatitis.

A better understanding of these objects and advantages of the present invention as well as the several steps and the relation of one or more of such steps with respect to each of the others and the features, properties and interactions of the compositions will be obtained from the following detailed description which sets forth a preferred embodiment of the invention and is indicative of the manner in which the principles of the invention are employed.

DESCRlPTlON OF A PREFERRED EMBODIMENT The diagnostic test method of the present invention generally involves the fractionation of human blood serum to be tested in such a manner as to concentrate a specific factor indigenous to hepatitis within a selected fraction, followed by the separation of that fraction and its preparation for both preliminary and confirmatory immunological test procedures. For best results the serum to be tested should be free of hemolysis or red blood cell stroma since such sera will give a weak positive reaction with the diagnostic test reagents. Such weak results are distinguishable from the results produced by hepatitis but might lead to some confusion. Accordingly, it is generally preferred that the blood to be tested be taken by venipuncture. Serum from such blood is obtained in the conventional manner and in accordance with the present invention is subjected to cryogenic cracking or fractionation which is believed to cause migration of the specific hepatitis factor to the bottom of the serum. A select glycinesaline processing reagent of particular concentration and pH is added to the cryogenic fraction after separation thereof and the resultant mixture is incubated for a short period. The incubated solution, referred to as the processed serum," is then employed in both the preliminary and confirmatory test procedures. ln the preliminary test procedure substantially equal portions of the processed serum and a special immunological diagnostic reagent are mixed on a glass slide. A positive reaction occurs within one minute and is clearly apparent from the formation of a heavy flocculent or colloidal aggregate which results when hepatitis is present. The confirmatory test procedure employs the same im munological reaction but utilizes a more refined and discriminatory technique employing precisely measured amounts of the processed serum and diagnostic reagent followed by intermixing and macroscopic reading of the sedimentation rate within the mixture.

SERUM FRACTIONATION As mentioned, it is imperative in the test procedure of the present invention that the serum to be tested be subjected to cryogenic fractionation prior to treatment with the diagnostic test reagents. The cryogenic fractionation of the serum takes place over a period of more than one hour and preferably about two hours or more to permit migration of the hepatitis factor and concentration thereof near the bottom of the test sample. In accordance with the preferred method the serum is placed in a clean dry test tube and inserted into a freezer having an ambient temperature well below the freezing temperature of the serum. In practice a temperature of approximately -20C has been found satisfactoryv The serum slowly solidifies and in so doing facilitates the downward migration or cryoprecipitation of the hepatitis factor. After a period of about 2 hours or more the test tube is removed from the freezer and permitted to come to room temperature without supplying additional heat or otherwise disturbing the relative positioning of the serum components. The serum will exhibit three or four readily discernible layers upon completion of the thawing process.

Although neither the invention nor any aspect thereof should be restricted to a particular theory of operation, a limited theoretical explanation will assist in the understanding of the invention. It is theorized that the elevated immunoglobulin concentration in the blood ofpatients having hepatitis results from a process which differs from other liver diseases. Additionally, it

is believed that persons who contract or are exposed to infectious or serum hepatitis will exhibit a separate globulin-binding factor in their serum, perhaps in the nature of a hapten. This factor will be found within 72 hours after exposure and is hereinafter referred to as the "hepatitis factor." it is believed to enzymatically react with the liver in the presence of an unknown accelerator so that the increase in immunoglobulin concentration in cases of hepatitis is not due solely to antibody production but is due at least in part to a replacement of the antibodies bindably reacting with the hepatitis factor.

The complex component resulting from the reaction of the hepatitis factor with the immunoglobulin tends to act in a manner similar to the globulins and during the freezing operation will migrate toward the bottom of the serum thereby effecting a cryogenic cracking or Stratification of the serum. It is clear that some downward travel of gamma globulin takes place upon freezing and it is theorized that by concentrating the globulins within a certain portion of the serum, the hepatitis factor will also be concentrated within that area. As mentioned, when the serum is slowly thawed it separates into a plurality of discernible layers or stratum which can be readily separated. It has been established by electrophoretic measurements that the greatest concentration of gamma globulin is in the bottom layer or cryoprecipitate' of a cryogenically fractionated serum. Additionally, positive and reproducible test results using the procedure of the present invention showed a percent positive test reaction with the bottom layer, while the middle layer achieved a 42.8 percent positive test result and the top layer exhibited only an ll.4 percent positive test reaction. it will, of course, be appreciated that the cryogenic cracking of the serum may also tend to isolate hepatitis factor inhibitors or other materials in the upper layers, thereby permitting the consistent and reproducible positive results from the bottom layer. This possibility is fortified by the false negative results exhibited by some samples of whole nonfractionated serum.

Recontamination of the bottom layer with a minute amount of one of the upper layers may result during separation of the three layers unless appropriate care is taken. In the event of slight contamination by a small percentage of the upper layer some false positive reactions can be expected. Studies have shown that approximately 2 percent false positive reactions may be attributed to such contamination. However, these false positive reactions can be eliminated by critically pipetting the bottom layer when separating the sera and by assuring complete cryogenic cracking of the sera, that is, by maintaining the sera in its frozen state for a longer period of time or by repeated fractionation. in this connection it has been found that leaving the serum in the freezer at 20C for at least 2 hours generally gives satisfactory results.

As an indication of the beneficial results of the cryogenic process of the present invention, the test of the present invention was carried out using sera from a control group clinically free of hepatitis. From this group there were 320 (32.5 percent] that gave weak positive reactions in the preliminary test procedure of the present invention when whole nonfractionated serum was utilized, With the exception of four cases (0.42 percent) these reactions were eliminated when the sera was processed in accordance with the cryogenic technique of the present invention. One of these four was hospitalized 34 days after the test was perfonned and a diagnosis of infectious hepatitis resulted from this hospitalization. The remaining three persons remained asymptomatic. When whole unfractionated sera of patients who were known cases of hepatitis was utilized, 4.1 percent of the control group gave negative test results. However, when the sera was cryogenically separated in accordance with the procedure of the present invention the sera of all of the hepatitis patients exhibited gross positive reactions. in this connection it should be noted that the reactions were not complement dependent since the complement was purposely deactivated by heating for one-half hour at 58C.

In keeping with the postulated theory regarding the migration of immunoglobulins during the cryogenic separation, electrophoretic determinations of the protein content of the sera were made on both the whole sera and on the cryogenically cracked material. With the cryogenically fractionated material the top layer of sera showed an increase in albumin while the middle layer showed a moderate increase in albumin and a slight increase in the alpha and beta protein fractions. The bottom layer or cryoprecipitate of the sera showed the greatest increase in the gamma globulin content with a decrease in the albumin fraction.

PROCESSING OF CRYOPRECIPITATE FRACTION As mentioned hereinbefore, the material to be subjected to the preliminary and confirmatory test procedures of the present invention is an incubated mixture comprised of the cryoprecipitate fraction and a specific processing solution. The mixture contains from about 6 to 9 percent cryoprecipitate and is allowed to incubate for a period of time sufficient to separate the hepatitis factor from the immunoglobulin for subsequent detection. Although the incubation period may vary slightly it has been found that a period of about l5 minutes is generally sufficient when the mixture is intermittently mixed or stirred. This incubation period has been studied by electrophoresis to provide a better understanding of the operative mechanisms of the procedure. As mentioned. the cryoprecipitate exhibits an increased gamma globulin content. However, after permitting a mixture of the cryoprecipitate and the processing solution to stand at room temperature for 5 minutes, it was shown by electrophoresis that there was a sharp decrease in the gamma globulin content. After permitting the mixture to stand at room temperature for fifteen minutes electrophoresis indicated a complete absence of all immunoglobulins with albumin being the only protein fraction remaining in the mixture. Thus, it is clear the incubated or processed serum used in the present invention contains no gamma globulin prior to the preliminary and confirmatory test procedures. Since the processed serum has consistently and reliably confirmed the presence of hepatitis in serum from patients known to have hepatitis, it is believed the hypothesis regarding the presence of a hepatitis factor is confirmed. Apparently, the factorglobulin complex of the serum is disassociated during the incubating phase of the processing. This disassociation liberates the hepatitis factor and allows it to react with the diagnostic reagent. The mechanism appears to be specific to cases of hepatitis and is not dependent on the presence of immunoglobulins within the serum.

The alkaline solution preferably employed in the processing of the serum fraction is an aqueous solution of glycine (aminoacetic acid) and sodium chloride having a pH of about 8.6-9.2. Best results have thus far been obtained at a glycine concentration level of about 0.l mole per liter and a sodium chloride concentration of about 0.3 mole per liter although the sodium chloride concentration may be slightly less, varying down to as little as about 0.2 mole per liter. The pH of the glycine-saline solution can be readily adjusted to the desired level with an 0.l molar sodium hydroxide solution and the solution generally also contains 0.l percent sodium azide as a preservative.

The cryoprecipitate is admixed with the glycinesaline processing reagent in proportions of from about l:l3 to about l:l7. These proportions may vary slightly so long as the desired gamma globulin denaturization is attained, the preferred proportion being about lzl6. As mentioned, the processed serum is used for the initial and confirmatory test procedure after incubation at room temperature for a period of about 15 minutes.

TEST REAGENTS The diagnostic test procedures of the present invention involve an immunological reaction between the hepatitis factor in the processed serum and a globulin fraction contained within the diagnostic reagent. The immunological reaction takes a form similar to conventional antigen-antibody systems but, because of the particle size of the colloidal aggregate produced by the reaction, requires the use of a carrier to expedite the macroscopic visualization of an otherwise invisible reaction. For the purpose of the present invention it has been found that latex particle carriers of substantially uniform size provide excellent results, giving an optimum degree of specificity and sensitivity coupled with improved speed and quality of reaction. Thus, in both the preliminary and confirmatory test procedures substantially the same diagnostic reagent is employed.

The improved diagnostic reagent of the present invention is an alkaline saline solution of latex particles coated with a gamma globulin fraction. Particles of synthetic organic resins in the form of a latex suspension have been found best suited for acting as carriers so long as the size and shape of the particles can be closely controlled. It is desirable to use a particle of uniform size and shape such as spheres having a diameter between about 0.5 and 1.0 micron. Suspensions of such particles can be obtained by the polymerization of the resin under controlled conditions. Particularly preferred are polystyrene particles having a generally spherical shape and a diameter of 0.8l micron with a deviation of only about 0.0063 micron. These spheres are believed to have a molecular weight in excess of 200,000 and to be negatively charged in suspension.

A stock solution of polystyrene latex particles is ob tained by appropriate dilution of the latex suspension with sterile distilled water. The degree of dilution will vary depending on the initial concentration of the suspension and is generally a l:l0 stock suspension. However, in accordance with the present invention the proper concentration is reached when a solution consisting of 0.l ml. of the stock solution and 10 ml. of water permits 5 percent light transmission through a sample 13 X 13 X 100 mm. as measured on a Coleman spectrophotometer (Model I l or 14) at 650 mp fitted with a red filter. Although the stock latex solution thus prepared may be stored in refrigeration for prolonged periods, it should not be frozen if best results are to be achieved.

The diagnostic reagent is an alkaline saline solution of the latex particles coated with human gamma globulin, such as Cohn Fraction ll obtained from human serum or plasma fractionated by methods 6 and 9 of Cohn and composed mainly though not exclusively of gamma globulin. The reagent is prepared by mixing the described stock latex solution with the gamma globulin in the presence of a suitable buffer. Accordingly, for this purpose there is prepared a glycine-saline buffer solution of somewhat different concentration from that used during the processing of the fractionated serum. This can be readily accomplished by adjusting the pH ofa 0.1 molar solution of glycine to 8.2 with lN sodium hydroxide and then dissolving slightly less than about 0.2 mole of sodium chloride therein.

A stock solution of the human gamma globulin fraction to be used as the reactant can be prepared using the above-described buffer solution. The reactant is a gamma globulin fraction previously incubated at about 57C for l5 to 30 minutes and is used in the form ofa l percent solution in the buffer. In accordance with the preferred embodiment of the present invention the reactant is the gamma globulin fraction II of pooled human sera, that is, Cohn Fraction ll. However, euglobulin or gamma globulin fraction ll from patients with rheumatoid arthritis may also be employed. Where the gamma globulin fraction takes the form of a solid, l gram thereof can be conveniently dissolved in lOO ml. of the buffer solution using 5-l0 ml. increments, the supernatant fluid from each increment being collected until the powder is completely dissolved. The solution may then be centrifuged and filtered to provide the desired l percent gamma globulin fraction ll stock solution.

The desired latex-globulin diagnostic reagent is ob tained by intermixing the stock solutions of polystyrene latex, gamma globulin reactant and saline buffer in suitable proportions. The latex solution and reactant solution are admixed in proportions of about lzS and then diluted by the addition of about 100 parts by volume of buffer to yield the final reagent formulation. This will result in a latex particle concentration sub stantially equal to that used in determining the proper dilution for the stock solution. The resultant concentration of the globulin fraction will be about 0.05 percent. About 0.l percent sodium azide may be added to the reagent as a preservative and the resultant solution stored under refrigeration.

TEST PROCEDURES The diagnostic test procedures utilized in accordance with the present invention include both preliminary and confirmatory testing operations. The preliminary test provides an extremely rapid determination of the possible existence of infectious or serum hepatitis while the confirmatory test procedure read macroscopically is only slightly more time consuming but far more discriminatory in its results.

The preliminary test merely involves the intermixing of equal portions, e.g., 0.05 ml. of both the processed serum and the latex-globulin diagnostic reagent on a glass slide. After gently rocking the slide back and forth for about I minute to assure interaction, the results are by transillumination of the slide. A positive reaction is characterized by gross agglutination, that is, the formation of visible colloidal aggregates of a regular and individual character as opposed to an irregular colloidal form best categorized as a gel. in the event of a positive reaction in the slide test, the confirmatory test should be performed.

False positive reactions may be obtained in the slide test from hemolyzed blood or serum containing red blood cell stroma. However, this type of reaction is markedly different from that produced by hepatitis. The reaction with stroma results in a loose flakey and irregular type of agglutination categorized as a gel as compared with the regularly formed colloidal aggregates indicating a positive test for hepatitis.

The confirmatory test procedure is carried out in a calibrated capillary reaction tube having a first scored marking at 0.03 ml. and a second marking at 0.07 ml. The latex-globulin reagent is first drawn into the capillary tube until it reaches the 0.03 mark. The processed serum is then drawn into the capillary using the same entrance end until the top of the diagnostic reagent reaches the 0.07 mark on the capillary. The solutions within the capillary are then intermixed by gently and repeatedly inverting the capillary tube 10 or more times to permit thorough interaction between the materials carrying charges of opposite polarity, i.e., the negatively charged polystyrene-globulin particles and the positively charged hepatitis factor. The tube is finally inverted and mounted within a layer of clay which serves to seal the end of the capillary in an electrically neutral medium while at the same time holding the tube erect for observation of the agglutination and sedimentation within the mixture. Agglutination or flocculation generally occurs within 15 to 20 minutes in the case of both infectious and serum hepatitis and the sedimentation rate can be readily followed over a time span of about 1 hour. During this time, readings are taken at frequent intervals such as every ten or fifteen minutes and any differences in the sedimentation rate are noted. The sedimentation rate for infectious and serum hepatitis is substantially different from that caused by hemolyzed sera. In fact, the sedimentation resulting in the case of hepatitis is generally complete l5 to 20 minutes sooner than that caused by hemolyzed sera. it has also been found that the confirmatory test method produces a negative result within one hour in all cases where the preliminary test gave weak false positive results due to cortisone therapy and red blood cell stroma.

The above-noted differences in sedimentation rate can be better appreciated by considering the dynamics of the system. It is known that if particles in an isodimensional suspension are allowed to settle of their own accord, they well tend to pack themselves in such a way that a sediment of minimum free energy results. This means that the particles will line up with their large surfaces lying opposite to each other. The

unchanged solvated layers or lyospheres will thereby act like a lubricant permitting the particles to slide over each other until they have found their proper locations. The polarity change resulting from the presence of the hepatitis factor and the resultant elimination of the polystyrene's self-repelling force permits the rate of sedimentation to be proportional to the mass of the colloidal aggregates involved. The neutralized particles will then tend to coalesce or cohere to each other to form colloidal aggregates which will result in comparatively loose packing and an acceleration in the sedimentation rate. Additionally, the solvated layers of the first settled particles are distorted by the pressure exerted upon them and this in turn assists the remaining particles in coming to rest more rapidly.

It will be appreciated that the combination of the styrene-globulin particles with the hepatitis factor will result in a primary colloidal particle of submicroscopic size exhibiting only discrete discontinuities. These primary colloidal particles then associate without losing their individuality to form the visual colloidal aggregates characteristic of a positive test reaction. However, in the case of hemolyzed sera or sera containing red blood cell stroma, the particles surpass the colloidal range and form a flaky, irregular agglutinate classified as a gel rather than a true colloidal aggregate. The visual difference between gel formation and colloidal aggregate formation is readily distinguishable particularly when coupled with the difference in sedimentation rate.

The test procedure of the present invention also provides for distinguishing between serum hepatitis and infectious hepatitis even though it is believed both are caused by the same etiologic agent. Hy carefully following the sedimentation rate in the confirmatory test procedure, it can be observed that all cases of infectious hepatitis exhibit a faster sedimentation rate than those of serum hepatitis. This difference is believed to be due to factors associated with the individual patients, such as the route of transmission, gamma globulin concentration and general condition of the liver as well as the medications received by individual patients.

Typical of the results achieved by utilizing the preliminary and confirmatory test procedures of the present invention are those set forth in greater detail in Table l with respect to a large number of related liver diseases.

TABLE I Lil ditia) 4 3 l r 3 I i O C infectious mononucleoais 24 2 l 3' 23 l obstructive jaundice 5 5 0 5 0 alcoholic cirrhosis 73 73 0 73 0 sickle cell anemia l 2 I 2 0 l 2 0 Cooley: anemia 3 3 O 3 0 weakly positive -patient was receiving cortisone therapy '6alcoholic cirrhosis 2-inaecticide poisoning l-tetrachloride poisoning 4-of unknown origin 4barbiturate overdose '"patient had hemolytic process, plasma hemoglobin was 0.06 grams As can be seen from Table 1 positive test results were achieved in percent of those cases having infec tious or serum hepatitis and negative results were obtained in about 99 percent of all cases of liver diseases other than serum and infectious hepatitis.

In order to better evaluate the test procedure, some tests were performed in conjunction with serum bilirubin and serum pyruvic transaminase (S.G.P.T.). The bilirubin concentration for cases of serum and infectious hepatitis ranged from 1.8 mg. percent to 19 mg. percent and the pyruvic transaminase values ranged from 34 units to L400 units. When the test procedures of the present invention were performed on these patients positive results were obtained up to the 16th month after the onset of hepatitis. Eleven cases of known hepatitis were followed on a monthly basis and the tests remained positive for an average of l 1 months after the onset of the disease.

The following examples are given in order that the effectiveness of the present invention may be more fully understood. These examples are set forth for the purpose of illustration only and are not intended to in any way limit the practice of the invention.

EXAMPLE I Approximately 8 mls. of human blood was drawn from a patient believed to have hepatitis, care being taken to avoid hemolysis by removing the needle from the syringe before placing the blood sample in a test tube. The blood sample was centrifuged at 1,800 rpm for [0 minutes and the supernatant serum was decanted into a clean dry test tube. The serum was placed in a freezer and allowed to remain therein for 2 hours, the freezer temperature being approximately -20C. After removal from the freezer the serum was permitted to completely thaw at room temperature without being stirred. After the serum had reached room temperature most of the thawed serum's bottom layer was carefully separated with a pipett and the top layers of the serum were discarded.

A processing reagent was prepared as follows: 7.8 grams of glycine and 17 grams of sodium chloride were dissolved in a liter of water and the pH of the solution was adjusted to 8.6-9.2 with a 10 percent sodium hydroxide solution. To this was added O.l percent sodium azide as a preservative.

About 0.8 ml. of the processing solution was pipetted into a clean dry test tube to which was added 0.05 ml. of the serum's bottom layer. The resultant mixture was allowed to stand at room temperature for an incubation period of IS minutes with mixing every minutes during the incubation period. The resultant product is hereinafter referred to as the processed serum.

Using sterile precautions, a latex gamma globulin diagnostic reagent was prepared from 0.5 ml. ofa 1:|0 stock latex solution, 0.5 ml. ofa stock 1 percent gamma globulin (Cohn Fraction ll) solution and mls. of an alkaline buffer solution. The latex and gamma globulin solutions were mixed and heated at 57C for to 30 minutes prior to the addition of the buffer.

The buffer solution was prepared from 975 mls. of an 0.1 molar glycine solution to which was added 2.5 mls. ofa lN sodium hydroxide solution. Water was added to bring the solution to l liter and the pH was adjusted to 8.2. To this was added l0 grams of sodium chloride to provide the stock buffer solution.

A polystyrene latex suspension of polystyrene particles having an average diameter of 0.8l micron was diluted with sterile distilled water until the solution exhibited 5 percent light transmission when appropriately diluted and measured on a spectrophotometer in the manner set forth hereinbefore. The stock solution was substantially a l:l0 solution of a polymer suspension having 3060 percent solids.

A stock I percent gamma globulin solution was prepared by dissolving one gram of lyophilized gamma globulin fraction ll of pooled human sera in 100 mls. of the stock buffer solution.

The preliminary test procedure was conducted as fol lows: About 0.05 ml. of the processed serum and 0.05 ml. of the diagnostic reagent were mixed on a slide with a glass rod. The slide was then gently rocked back and forth for a period of about I minute. Transillumination of the slide revealed gross agglutination within the mixture indicative ofa positive reaction for hepatitis.

To confirm the test results a scored capillary reaction tube was filled with the diagnostic reagent to the 0.03 ml. mark. Using the same end ofthe capillary tube, the processed serum was drawn into the tube until the re agent reached the 0.07 ml. mark on the tube. The solutions within the capillary tube were gently mixed by tilting the tube approximately 10 times after which the tube was inverted and placed in an upright position within a layer of clay. Within minutes the solution evidenced gross agglutination indicating a positive reaction for infectious hepatitis.

EXAMPLE II A control group of patients previously determined to be clinically negative for hepatitis were tested in accordance with the test procedure of the present invention using procedures and reagents substantially identical to those set forth in Example I. The total number of sera tested were 1,879 From this number, 24 or L3 percent positive test reactions were obtained. From among these 24, I0 were found to have hepatitis and four were asymptomatic but are being followed. lt was not possible to follow the remaining 10 cases.

l EXAMPLES Ill Another group of 1,425 patients were tested in a blind study using the reagents and procedures substantially identical to those set forth in Example I. The only information supplied prior to testing was that these patients had liver diseases. From among this group positive test results were obtained on 3l (5.5 percent) paiients. It was later disclosed that these 3| cases were the total number of known hepatitis cases placed in the blind study group as a control.

Another blind study group consisting of 1,848 patients were tested in accordance with the test procedure of the present invention. This group consisted of 983 normal patients, 675 patients with diseases other than serum or infectious hepatitis and known cases of hepatitis. When tested in accordance with the present invention, I89 hepatitis cases were positively identified and four of the normal group were also found to give positive results.

EXAMPLE IV In another selected study, 190 known hepatitis cases were tested in the manner set forth in Example I. All 190 ([00 percent) of the cases gave positive test reactions in both the preliminary and confirmatory test procedures.

In one field study, 750 patients were tested in accordance with the test procedure of the present invention. From among these, 1 l were known cases of hepatitis. The test procedure of the present invention detected all I 1 known cases.

In a separate field study of blood donors, 2l (2.4 percent) of the sera tested gave positive results from the test procedure of the present invention. Two of those indicating positive results have since been found to have hepatitis. The other l9 are being followed.

Sera from patients who had undergone surgical procedures using extracorporeal circulation were found to give false positive reactions of the gel type for about the first two weeks after surgery. ln this group 93 percent (465 out of 489) gave a gel type of agglutination. The average plasma (hemoglobin) of these patients was 0.08 gram percent. The patients in this group received either whole blood alone, whole blood and gamma globulin or whole blood, gamma globulin and fibrinogen. The false positive results can be grouped as follows: (l) patients receiving 2 units or more of whole blood reacted up to the llth day; (2) patients receiving whole blood and gamma globulin reacted up to the lSth day; and (3) patients receiving whole blood, gamma globulin and fibrinogen reacted up to the l7th day. In all cases of extracorporeal circulation surgical patients, except five, sera gave negative results after the above-stated periods. In the five cases giving longer reactions, three were later admitted to the hospital with diagnoses of serum hepatitis.

As can be seen from the foregoing detailed description, the present invention provides an extremely reliable and highly selective test procedure for the detection of both infectious and serum hepatitis. it enables a clinician to rapidly differentiate between hepatitis and other liver diseases while at the same time obviating a high percentage of nonspecific reactions of a false negative or false positive nature including those caused by factors such as hemolyzed blood, blood containing red blood cell stroma, blood from patients receiving cortisone therapy and blood from patients having recently undergone extracorporeal circulatory procedures. The procedure of the present invention also provides for the isolation of a specific hepatitis factor and utilizes a specific diagnostic reagent which visibly demonstrates the presence of the hepatitis factor within human blood serum. The diagnostic test procedure of the present invention is also effective in producing positive test results from one month before the onset of hepatitis up to about twelve months after the occurrence of the disease.

As will be apparent to persons skilled in the art, various modifications, adaptations and variations of the foregoing specific disclosure can be made without departing from the teachings of the present invention.

I claim:

I. A serological test procedure for the immunological detection of infectious or serum hepatitis in human blood serum comprising the steps of lowering the temperature of said human blood serum below its freezing point to cause migration of at least a portion of the globulin content therein to the lowermost portion of the serum and to effect concentration of a hepatitis factor within said lowermost portion, thereafter thawing the frozen serum to produce discernible layers within the serum, the lowermost fluid serum layer having concentrated hepatitis factor therein, separating said lowermost layer from the remainder of the serum, mixing said layer containing the concentrated hepatitis factor with a glycine-saline solution having a pH of about 8.6 to 9.2 and a salt concentration of about 0.20.3 mole per liter for a period of time sufficient to provide a processed serum substantially free of gamma globulin, and thereafter bringing the processed serum into intimate contact with an agglutinating reagent compris ing human gamma globulin coated on latex carrier particles for forming macroscopic colloidal aggregates indicative of a positive reaction for infectious or serum I4 hepatitis.

2. The test procedure of claim 1 wherein the human blood serum is cooled to an ambient temperature of about 20C for a total lapsed period of at least 1 hour.

3. The test procedure of claim 1 wherein the human blood serum is cooled to an ambient temperature of about -20"C for about 2 hours, and the glycine-saline solution has a salt concentration of at least about 0.23 mole per liter, the latex carrier particles are polystyrene particles of about 0.81 micron and the concentration of the human gamma globulin is about 0.05 percent by weight.

4. The test procedure of claim 1 wherein the proportion of serum layer to saline solution is from about l:l3 to about H7.

5. The test procedure of claim 1 wherein the latex carrier particles are polystyrene particles and the agglutinating reagent includes a glycine-saline bufi'er solution having a pH of about 8.2 and a salt concentration of less than about 0.2 mole per liter.

6. The test procedure of claim 1 wherein the processed serum and the agglutinating reagent are mixed in approximately equal proportions on a slide.

7. The test procedure of claim I wherein the processed serum and the agglutinatin reagent are mixed withln a reaction vesse In propo IOI'IS of about 8. The test procedure of claim 1 including the step of observing the sedimentation rate of the macroscopic colloidal aggregates resulting from the reaction.

9. The test procedure of claim 1 wherein the human blood serum is cooled to an ambient temperature of about 20C for about 2 hours, and the glycine-saline solution has a salt concentration of at least about 0.23 mole per liter.

10. The test procedure of claim 9 wherein the proportion of serum layer to saline solution is from about l:l6, the sodium chloride concentration is about 0.3 mole per liter and the glycine concentration is about 0.1 mole per liter. 

2. The test procedure of claim 1 wherein the human blood serum is cooled to an ambient temperature of about -20*C for a total lapsed period of at least 1 hour.
 3. The test procedure of claim 1 wherein the human blood serum is cooled to an ambient temperature of about -20*C for about 2 hours, and the glycine-saline solution has a salt concentration of at least about 0.23 mole per liter, the latex carrier particles are polystyrene particles of about 0.81 micron and the concentration of the human gamma globulin is about 0.05 percent by weight.
 4. The test procedure of claim 1 wherein the proportion of serum layer to saline solution is from about 1:13 to about 1:17.
 5. The test procedure of claim 1 wherein the latex carrier particles are polystyrene particles and the agglutinating reagent includes a glycine-saline buffer solution having a pH of about 8.2 and a salt concentration of less than about 0.2 mole per liter.
 6. The test procedure of claim 1 wherein the processed serum and the agglutinating reagent are mixed in approximately equal proportions on a slide.
 7. The test procedure of claim 1 wherein the processed serum and the agglutinating reagent are mixed within a reaction vessel in proportions of about 4:3.
 8. The test procedure of claim 1 including the step of observing the sedimentation rate of the macroscopic colloidal aggregates resulting from the reaction.
 9. The test procedure of claim 1 wherein the human blood serum is cooled to an ambient temperature of about -20*C for about 2 hours, and the glycine-saline solution has a salt concentration of at least about 0.23 mole per liter.
 10. The teSt procedure of claim 9 wherein the proportion of serum layer to saline solution is from about 1:16, the sodium chloride concentration is about 0.3 mole per liter and the glycine concentration is about 0.1 mole per liter. 